Genetic manipulation of the pancreas: cell and gene therapy approaches for type 1 diabetes

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(1)FACULTAT DE VETERINÀRIA CENTRE DE BIOTECNOLOGIA ANIMAL I TERAPIA GÈNICA (CBATEG). GENETIC MANIPULATION OF THE PANCREAS: CELL AND GENE THERAPY APPROACHES FOR TYPE 1 DIABETES. EDUARD AYUSO LÓPEZ.

(2) Memòria presentada pel llicenciat EDUARD AYUSO LÓPEZ per optar al grau de Doctor en Bioquímica.. Aquesta Tesi Doctoral ha estat realitzada sota la direcció de la Dra. Fàtima Bosch i Tubert en el Departament de Bioquímica i Biologia Molecular de la Facultat de Veterinària i al Centre de Biotecnologia. Animal i. Terapia. Gènica. (CBATEG).. EDUARD AYUSO LÓPEZ. FÀTIMA BOSCH I TUBERT. MAIG de 2006 BELLATERRA.

(3) A mis padres, y a mi hermano, por aportar toda la estabilidad, confianza y ayuda incondicional imprescindible para realizar este trabajo.. A Sabrina, por todo lo que hemos superado juntos, y por todo lo que nos espera en el presente y el futuro..

(4) Abbreviations. ABBREVIATIONS. AAV ABC Ad ADP AMPK ATP BAD BM BMC bp BSA ß-gal cAMP CAR cDNA CAG Ci CMV Con cpm DAB DAG dATP DCCT dCTP DMSO DNA Dnase dNTPs ds EDTA Fas FasL FBS FG-Ad FITC g GAD GFP GH. Adeno-asociated virus Avidin-biotin complex acid Adenovirus Adenosine-5'-diphosphate AMP-activated protein kinase Adenosine-5'-triphosphate Pro-apoptotic protein Bone marrow Bone marrow cells base pair Bovine Serum Albumin ß-galactosidase cyclic adenosine-5'-monophosphate Coxsackie virus adenovirus receptor copied DNA cytomegalovirus enhancer and chicken beta actin hybrid promoter curie cytomegalovirus control counts per minute 3’3’-diaminobenzidine diacylglycerol desoxiadenosin-5'-triphosphate diabetes control and complications trial desoxicitidin-5'-triphosphate dimethyl sulfoxide Desoxiribonucleic acid deoxyribonuclease 2'-deoxynucleosides 5'-triphosphate double stranded ethylenediamine-tetraacetic acid Fas, cell death receptor Fas ligand Fetal Bovine Serum First generation adenoviral vectors fuorecein isothiocyanate grams Glutamic acid decarboxilase Green fluorescein protein Growth hormone.

(5) Abbreviations GK GLP-1 h hCMV HC-Ad HD-Ad HEK HIV HLA HPRT IFN- IFN/IGF-I IGFBP IGF-I IGF-II IGF-IR IL IM iNOS Ip IP3 IR IRS-1 IRS-2 IU Kb KDa KO M MAPK MCS mg MHC min ml MLD mM mRNA NO NOD o/n OD 32 P PBS PCR PDX-1. glucokinase glucagon-like peptide 1 hour human cytomegalovirus High capacity adenoviral vectors Helper-dependent adenoviral vectors Human Embryo Kidney human immunodeficiency virus Human leukocyte antigen Hypoxanthine-guanine phosphoribosyltransferase beta interferon double transgenic mice RIP/IFN and RIP/IGF-I Insulin-like growth factor binding protein Insulin-like growth factor-I Insulin-like growth factor-II Insulin-like growth factor receptor-I interleukine Intramuscular inducible nitric oxide sinthase intraperitoneal inositol triphosfphate Insulin receptor Insulin receptor substrate 1 Insulin receptor substrate 2 infection units of adenoviral particles Kilobase kiloDalton knock-out molar (mol/l) mitogen-activated protein kinase Multiple Cloning Site milligrams major histocompatibility complex minute/s millilitre multiple low dosis millimolar RNA messenger Nitric oxide non obese diabetic over night optical density Phosphor 32 Phosphate-Buffered Saline Polymerase Chain Reaction pancreatic duodenal homeobox gene.

(6) Abbreviations PI-3K PKB pp r.t. RIP/ hIFN RIP/ IGF-I RIP-I RIP-II RNA RNase rpm SDS sec SEM Ss SSC SMA STZ Tg TRITC UV vol vg WPRE xg. phosphatidyl inositol 3 kinase protein kinase B Physical particles room temperature Rat insulin promoter-I– cDNA human IFN Rat insulin promoter-I– cDNA murine IGF-I Rat insulin promoter-I Rat insulin promoter-II ribonucleic acid ribonuclease revolutions per minute Sodium Dodecyl Sulphate seconds standard error from the mean single stranded sodium chloride and sodium citrate solution smooth muscle actin streptozotocine (2-deoxi-2-(3-metil-3-nitrosourea)1Ddlucopyranose transgenic texas red isothiocyanate Ultraviolet volum vector genomes Woodchuck post-transcriptional regulatory element gravity unit.

(7) AGRADECIMIENTOS Son muchos los años que llevo en este grupo y por tanto muchas son las personas que han contribuido en este trabajo. Además, yo siempre he creído que nosotros somos la suma de las personas que hemos conocido a lo largo de nuestra vida, y de ahí que todas estas personas son importantes para mí. En primer lugar a la Dra Fàtima Bosch, por ser mi profesora de bioquímica, y así iniciarme en este apasionante campo, y sobretodo agradecer la oportunidad que me dió para incorporarme a su grupo desde el año 96 hasta hoy en día. Siempre ha confiado en mí y me enseñado lo que significa trabajar en ciencia. Por su dedicación al trabajo y por hacer crecer la investigación en nuestro país. A Judith, por su enorme contribución profesional a esta tesi y por su aportación personal que ha marcado una etapa de mi vida. A Mónica, que supo sacar lo mejor de mi, darme la motivación necesaria para continuar en este campo, y porque fué un placer trabajar juntos. Aunque el tiempo pasa me parece que siempre es el primer día que la conocí. A Vero, que desde su incorporación siempre ha estado a mi lado, ha trabajado muy duro y muy bien, y por ello le entrego mi confianza. A Marta, porque hemos crecido juntos, por hacer un trabajo impecable con las immunos, pero sobretodo por su carácter y poner siempre buena cara cuando voy a hablar contigo, aún sabiendo que no te espera nada bueno . Muchísimas gracias. A Alba, siempre a sido un placer trabajar con ella y discutir resultados, pero sobretodo tengo que agradecer su calidad humana, su disponibilidad, y su enorme amabilidad. A Ariana, que con su inagotable energía es una pieza clave del ‘pancreas club’ y siempre esta dispuesta a echar una mano. A Miguel Chillón, porque recuerdo con suma alegría cuando trabajábamos y diseñábamos juntos y porque me has enseñado tantísimo. A Pedro Otaegui, porque sigue en el ojo del huracán, por los animados debates que proporciona al grupo, y por su ayuda con los roedores. A Virginia, por el impulso que supuso su llegada, y por aportar valiosas discusiones científicas. A Jesús Ruberte y todo el grupo de morfología (Ana, Marc, Cris, Clara, Vero y Víctor) por la fructífera colaboración establecida. A Félix García y Ana Andaluz por su imprescindible ayuda en las cirugías. A Juan Bueren y Jose Carlos Segovia por su colaboración en los estudios de transplante de médula ósea..

(8) I would like to thank Stefan Kochanek for accepting me in his lab. Florian Kreppel for teaching me gutless production. Voglio ringraziare Mauro Giacca per avermi accettato nel suo laboratorio e per avermi trattato sempre come uno del suo gruppo. A Carles Ros, que siempre hace lo imposible para que no nos falte de nada, por su buen humor y por enriquecer el diccionario de la real academia. Anna Vilalta por su ayuda técnica en veterinaria. Maria Molas, Jennifer, Lidia, y Mireia por ayudarme con células, virus, reactivos, ratones, y mucho más… Anna Pujol, Anna Arbós, y Ainara, por hacer funcionar la UAT. A Mercè Monfar, por ser tan ordenada y meticulosa, y por tener la paciencia de enseñarme. A Tura, porque admiro su capacidad de hablar y trabajar a vez, porque no se le escapa ningún detalle, y porque siempre ha estado y está dispuesta a ayudar. A Assumpció Bosch por compartir tus conocimientos y tu ayuda. A Maria Ontiveros, Jose y Susana que me echaron siempre una mano en la produccion de los vectors. Y hago extensivo este agradecimiento a toda la 5ª planta. A la apisonadora Àlex, y lo digo por su brutal capacidad, volumen de trabajo y su tesón, por haber compartido buenos momentos, y por todos los altibajos. Porque le aprecio y le deseo lo mejor. A Sergio, por saber compartir sus emociones en silencio, por hablar por los codos en otras ocasiones, por saber escuchar y saber escribir. A Sylvie, por aportar raciocinio científico al laboratorio, por saber vivir, y por compartir vila lucy con nosotros. A Miquel García, por ser casi un filósofo de la bioquímica, y por compartir sufrimientos en la 4ª planta escribiendo la tesis. A Antonio, porque hemos compartido tantas emociones, y por ser un verdadero COMPAÑERO en este y otros viajes. A Joel, por su calmado temperamento y su eficiencia en el trabajo, y por compartir alegrías y disgustos con los AAV. I would like to thank Jean Christoph for the heritage you left us on islet technology, cloning and PCR. My French-brother Laurent, thanks for all the time we spent together and for sharing with me all your experience. See you soon!.

(9) Poco a poco fueron llegando los más nuevos…Xavi, Carles, Ivet, Albert, Pilar, Gor, Gloria, Albert R., Andrés, ChrisMan y Sr. Callejas. Solo puedo decir que gracias a todos vosotros el ambiente del laboratorio no podría ser mejor, y el buen humor invade tanto el horario de trabajo como el de las comidas. Gracias a todos. Quiero destacar la ayuda prestada por Chris Mann y Malcom Watford para la corrección lingüística de esta tesis. Hannelore, Gustavo, Erika, Judith, and Huan Bin for making my stay at Ulm University very enjoyable. Lorena e Marina per il loro aiuto con i virus. A tutti voi, per il piacere di avervi conosciuto: Serena, Lucia, Sara, Chiara (piccola e grande), Lara, Marina, Barbara, Alejandro, Gian Luca, Alessandro, Paolo, Ana, Marlene, Ramiro, Federico, Bacir,… un grande saluto a tutto il ICGEB di Trieste. A Joseph García, que empezamos juntos en esto y vivimos tantos momentos intensos durante tantos años, por ser un gran amigo y ahora por ser un gran veterinario.. Agradezco a todo el personal de administración de Veterinaria (Fede, Carme, Silvia y Rosa) y del CBATEG (Montse, Espe, Esther, Mª José, Teresa, Lluís, Alba), y también el personal de seguridad porque su trabajo es imprescindible. Así mismo, por el trato cordial y por la labor que realizan en el departamento Joaquim Ariño, Anna Bassols, Anna Barceló y Nestor Gómez. También dedico un saludo a todos los miembros de sus respectivos grupos, por haber compartido con ellos el pasillo de veterinaria y tantos buenos momentos.. Este trabajo no hubiera sido posible sin la Beca de Formación de Personal Investigador (FI2002), Borsa Viatge (BV2004) y Beca per a Estades per a la recerca fora de Catalunya (BE2005) del Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya y del Fons Social Europeu. Las ayudas económicas nacionales del Instituto de Salud Carlos III (FIS01/0427, Red Grupos Diabetes Mellitus G03/212 y Red Centros Metabolismo y Nutrición C03/08), el Plan Naciona I+D+I (SAF2002-20389 y SAF2005-01262), La Fundació Marató de TV3 (992710), y las ayudas de la Generalitat de Catalunya al Grups de Recerca Consolidats (2001SGR00195 y 2005SGR00673); e internacionales, FP5 EuroDiabetesGene QLG1CT-1999-00674, FP6 EUGENE2 (LSHM-CT-2004-512013) y BetaCellTherapy ( FP62004-512145)..

(10) Thesis content Thesis Contents 1. I. SUMMARY II. INTRODUCTION 1. STRUCTURE AND FUNCTION OF THE PANCREAS. 3. 2. TYPE 1 DIABETES. 5. 3. CURRENT THERAPY FOR TYPE 1 DIABETES. 7. 3.1. Insulin replacement. 7. 3.2. Pancreas or islet transplantation. 8. 3.2.1. Pancreas transplantation. 8. 3.2.2. Islet transplantation. 9. 4. CELL THERAPY. 11. 4.1. Cell therapy for type 1 diabetes. 11. 4.2. Stem cells. 11. 4.2.1. Embryonic stem cells. 12. 4.2.2 Adult stem cells. 13. 4.2.2.1. Adult stem cells isolated from the pancreas, liver or intestine. 13. 4.2.2.2. Bone marrow stem cells. 14. 5. GENE THERAPY. 18. 5.1. Introduction to gene therapy. 18. 5.2. Viral vectors. 18. 5.2.1. Adenoviral vectors. 21. 5.2.1.1. Adenovirus structure and biology. 21. 5.2.1.2. First and second generation adenoviral vectors. 24. 5.2.1.3. Helper dependent adenoviral vectors. 25. 5.2.2. Adeno-associated viral vectors. 29. 6. GENE THERAPY FOR TYPE 1 DIABETES. 31. 6.1. Gene therapies aimed at reducing reactions or inducing immune tolerance. 32. 6.2. Genetic manipulation of extra-pancreatic cells to produce insulin. 33. 6.3. Gene transfer to the pancreas and/or -cells and its applications. 33. i.

(11) Thesis content 6.3.1. Gene transfer to pancreatic islets in vitro. 34. 6.3.2. Gene transfer to the pancreas in vivo. 36. 6.3.2.1. Direct injection to the parenchyma. 37. 6.3.2.2. Retrograde injection through the common bile duct. 38. 7. INSULIN-LIKE GROWTH FACTOR-I (IGF-I). 41. 7.1. IGF-I structure and function. 41. 7.2. Transgenic mice expressing IGF-I in -cells. 43. III. OBJECTIVES. 46. IV. RESULTS AND DISCUSSION. 47. PART I: CHARACTERIZATION OF BONE MARROW-DERIVED CELL FATE IN THE PANCREAS Introduction. 47. 1.BONE MARROW CELL TRANSPLANTATION IN TRANGENE MICE OVEREXPRESSING IGF-1 IN -CELLS. 49. 2. COUNTERACTION OF DIABETES IN BM-TRANSPLANTED IGF-I DIABETIC MICE. 51. 3. CONTRIBUTION OF BMC TO ENDOCRINE PANCREAS. 53. 4. CONTRIBUTION OF BMC TO EXOCRINE PANCREAS. 56. Discussion. 59. PART II: IN VIVO GENE TRANSFER TO MURINE AND CANINE PANCREAS Introduction. 64. 1. IN VIVO GENE TRANSFER TO MURINE -CELLS BY USING VIRAL 66. VECTORS 1. 1. Gene transfer to murine pancreas in vivo using first generation adenoviral vectors 1.1.1. Experimental design used for adenovirus administration to mice. 66 66. 1.1.2. Quantification of adenovirus in the bloodstream with and without portal clamp 1.1.3. Gene transfer to the pancreas in vivo. 67 68. ii.

(12) Thesis content 1.1.4. Characterization of pancreas transduction. 70. 1.1.4.1. Transduction of endocrine pancreas. 70. 1.1.4.2. Transduction of exocrine pancreas. 73. 1.1.5. Specific expression of the gene of interest in -cells using the rat insulin promoter. 76. 1.1.5.1. Generation of adenoviral vectors carrying RIP-II/-gal. 76. 1.1.5.2. In vivo administration of adenoviral vectors expressing -gal under the control of the rat insulin promoter. 80. 1.2. Gene transfer to murine pancreas in vivo using helper-depemdent adenoviral vectors. 81. 1.2.1. Production of helper-dependent adenoviral vectors carrying marker genes. 82. 1.2.2. In vivo administration of helper-dependent adenoviral vectors carrying marker genes. 83. 1.3. Gene transfer to murine pancreas in vivo using adeno-associated viral vectors and pancreas. 85. 1.3.1. Production of AAV vectors carrying marker genes. 85. 1.3.2. Comparison of different routes of administration for pancreatic gene transfer using AAV vectors. 87. 1.3.2.1. Intravascular delivery. 87. 1.3.2.2. Intraperitoneal delivery. 89. 1.3.2.3. Intraductal delivery. 91. 2. GENE TRANSFER OF IGF-I TO THE PANCREAS IN VIVO USING VIRAL VECTORS. 94. 2.1. Generation of IGF-1 expressing viral vectors. 94. 2.1.1. Generation and testing IGF-I-expressing helper-dependent vectors. 95. 2.1.2. Generation and testing IGF-I-expressing AAV8 vectors. 96. 2.2. In vivo administration of IGF-I expressing viral vectors into the pancreas. 98. 3. IN VIVO GENE TRANSFER TO HEALTHY AND DIABETIC CANINE PANCREAS. 99. 3.1. Experimental design used for adenovirus administration to canine pancreas 99 iii.

(13) Thesis content 3.2. Transduction of the pancreas. 101. 3.3. Transduced cell types. 103. 3.4. Evaluation of pancreatic and liver damage. 107. 3.5. Gene transfer to diabetic canine pancreas. 108. Discussion. 110. V. CONCLUSIONS. 118. VI. MATERIALS AND METHODS. 121. 1. ANIMALS. 121. 2. BONE MARROW TRANSPLANTATION. 121. 3. DIABETES INDUCTION. 122. 4. DNA BASIC TECHNIQUES. 123. 4.1. Bacterial culture. 123. 4.2. Plasmid DNA preparation. 123. 4.3. DNA enzymatic modifications. 123. 4.3.1. DNA digestion with restriction enzymes. 123. 4.3.2. Dephosphorylation of DNA fragments. 124. 4.3.3. Generation of blunt ends in DNA fragments. 124. 4.3.4. Ligation of DNA fragments. 124. 4.4. DNA resolution and purification. 125. 4.5. Transformation of competent E. coli. 125. 4.6. Construction of plasmids to generate viral vectors. 126. 4.6.1. Construction of pKP1.4 RIP-II/ß-gal. 126. 4.6.2. Construction of the HD-Ad vector expressing IGF-I. 127. 4.6.3. Construction of the AAV RIP-II/GFP. 128. 5. PRODUCTION, PURIFICATION AND CHARACTERIZATION OF FIRSTGENERATION ADENOVIRAL VECTORS. 129. 6. PRODUCTION, PURIFICATION AND CHARACTERIZATION OF HELPER DEPENDENT ADENOVIRAL VECTORS 6.1. Production and purification. 131 131. iv.

(14) Thesis content 6.2. Slot blot titration. 133. 6.2.1. Radioactive labelling of DNA probes. 134. 6.2.2. Prehybridization, hybridization and quantification. 134. 7. PRODUCTION, PURIFICATION AND CHARACTERIZATION OF AAV VECTORS. 135. 7.1. Production and purification. 135. 7.2. Slot blot titration of AAV vectors. 136. 7.2.1. Viral DNA extraction by proteinase K digestion. 136. 7.2.2. Transfer of DNA to the membrane, hybridization and quantification. 137. 8. RNA ANALYSIS. 138. 9. IN VIVO ADMINISTRATION OF VIRAL VECTORS IN MICE. 138. 10. LOCAL PANCREATIC CLAMP AND ADMINISTRATION OF ADENOVIRUS IN DOGS. 139. 11. MEASUREMENT OF ADENOVIRUS CONCENTRATION IN BLOOD. 140. 12. DETERMINATION OF SERUM PARAMETERS. 140. 13. ANALYSIS OF ß-GALACTOSIDASE EXPRESSION IN TISSUE SAMPLES. 141. 14. INCIDENCE AND SEVERITY OF INSULITIS. 141. 15. IMMUNOHISTOCHEMICAL ANALYSIS. 141. 16. MORPHOMETRICAL ANALYSIS. 142. 17. STATISTICAL ANALYSIS. 143. VII. REFERENCES. 144. VIII. ANNEX I IX. ANNEX II. v.

(15) I. SUMMARY.

(16) Summary. Type 1 diabetes is characterized by progressive destruction of pancreatic -cells, resulting in insulin deficiency and hyperglycemia. Insulin replacement therapy allows diabetic patients to lead active lives, but this therapy is imperfect and does not prevent development of severe secondary complications. Transplantation of pancreatic tissue or islets has been performed successfully in a limited numbers of patients. However, the shortage of donors is a primary obstacle that prevents this treatment from becoming more widespread. Therefore, many efforts have been focused on differentiating embryonic or adult stem cells into ß-cells. Bone marrow cells (BMCs) are an important source of easily procurable adult stem cells and have been proposed as an alternative source of ß-cells. Insulin-like growth factor-I (IGF-I) participates in skeletal muscle regeneration and enhances the recruitment of BMCs at the sites of muscle injury. In addition, IGF-I expression in ß-cells of diabetic transgenic mice regenerates pancreatic ßcell mass. Therefore one of the objectives of this study was to investigate whether IGFI expression in ß-cells could increase BMC recruitment and differentiation into ß-cells under steady-state conditions or after STZ treatment. To this end, BMCs from ßactin/GFP transgenic donor mice were transplanted into IGF-I transgenic mice. Our experiments have demonstrated that IGF-I overexpression or STZ-induced pancreatic damage were not sufficient to recruit and differentiate GFP-labelled BMCs into ß-cells in vivo, indicating that these cells did not contribute to the endocrine pancreas regeneration observed in IGF-I transgenic mice. These data suggest that replication of pre-existing ß-cells and/or differentiation from non-BMC precursors is the most likely mechanism for IGF-I-mediated regeneration.. Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with gene therapy. Recovery from type 1 diabetes requires ß-cell regeneration. One approach to do so is by genetically engineering the endocrine pancreas in vivo to express factors that induce ß-cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to the pancreas in vivo are required. In this study we have 1.

(17) Summary examined different viral vectors and routes of administration in rodents and also in large animals, to determine the most efficient method to deliver exogenous genes to the pancreas. First, we observed that pancreatic ß-cells were efficiently transduced to express ß-galactosidase after systemic injection of adenoviral vectors in mice with clamped hepatic circulation. This was true both for first generation as well as for helperdependent adenoviral vectors. In addition to adenoviruses, we have compared the ability of AAV vectors to transduce the pancreas in vivo after intravascular, intraperitoneal or intraductal delivery, being the last the most efficient route of administration. Like the human pancreas, the canine pancreas is compact, with similar vascularization and lobular structure. It is therefore a suitable model in which to assess gene transfer strategies. Here we examined the ability of adenoviral vectors to transfer genes into the pancreas of dogs in which pancreatic circulation has been clamped. Adenoviruses carrying the ß-galactosidase (ß-gal) gene were injected into the pancreaticduodenal vein and the clamp was released 10 min later. These dogs showed ß-galpositive cells throughout the pancreas, with no evidence of pancreatic damage. ß-gal was expressed mainly in acinar cells, but also in ducts and islets. ß-gal expression in the exocrine pancreas of a diabetic dog was also found to be similar to that observed in healthy dogs.. Thus, the methodology described herein may be used to transfer genes of interest to murine and canine pancreas in vivo, both for the study of islet biology and to develop new gene therapy approaches for diabetes mellitus and other pancreatic disorders.. 2.

(18) II. INTRODUCTION.

(19) Introduction 1. STRUCTURE AND FUNCTION OF THE PANCREAS.. The embryonic pancreas in vertebrates forms from a dorsal and ventral protrusion of the primitive gut epithelium. These two pancreatic buds grow, branch and ultimately fuse to form the definitive pancreas. The exocrine pancreas consists of acinar cells that produce and secrete a variety of digestive enzymes such as proteases, lipases and nucleases (Fig. 1). Another component of the exocrine pancreas is the highly branched ductal epithelium, which transports the digestive enzymes and bicarbonate ions to the intestine, where they contribute to the digestion of food. The endocrine pancreas constitutes about 2-3% of the total gland, and consists of four cell types -, -, -, and pancreatic-polypeptide cells that produce the hormones glucagon, insulin, somatostatin and pancreatic polypeptide, respectively (Fig. 1). Endocrine cells form clusters, called islets of Langerhans, and they are scattered irregularly throughout the exocrine parenchyma, most densely in the tail region. The ß-cells form a core that is surrounded by the other cell types. Although the bulk of the pancreatic endocrine tissue is organized as islets, single extrainsular endocrine cells can occasionally be found (Rahier et al., 1981). Of the endocrine cells, ~60–80% are insulin-producing ß-cells, 15–20% are glucagon-producing -cells, 5–10% are somatostatin-producing -cells and <2% are pancreatic-polypeptide-producing cells (Fig. 1).. Insulin is released from ß-cells in response to increased levels of blood sugar after food intake; this is a signal for the target tissues (liver, muscle and fat) to take up glucose. In addition, insulin inhibits glucose production in the liver. By contrast, glucagon secretion is stimulated at low blood-sugar levels. Glucagon promotes glycogenolysis and gluconeogenesis as the level of glucose in the blood decreases.. 3.

(20) Introduction Somatostatin and pancreatic polypeptide exert inhibitory effects on both pancreatic endocrine and exocrine secretion.. A. B. C. Figure 1. The pancreas is a mixed exocrine and endocrine organ. a) The mature pancreas is adjacent to the duodenum, the most anterior part of the small intestine. b) The function of the exocrine pancreas is to supply the gut with digestive enzymes; these are produced and secreted by acinar cells and subsequently transported to the intestine via the pancreatic ductal system. c) Endocrine pancreas consists of four hormone producing cell types -, -, -, and pancreatic polypeptide (PP) cells.. There are several differences between the rodent pancreas and large animals and humans. In rodents, the pancreas is a diffuse organ, whereas in large animal and humans it is compact. In the dog, it has the classic V-shape, consisting of two lobes (right and left) that emerge from the pancreatic body, surrounded by a delicate capsule of connective tissue. Septa from the capsule divide the pancreas into lobules, delimited by connective tissue, which produce a nodular surface with irregular crenate margins. Between the lobules, connective tissue surrounds the larger ducts, blood vessels, and nerve fibers. Branches from the celiac and the cranial mesenteric arteries supply blood to the pancreas. The pancreatic branches of the splenic artery irrigate the left lobe,. 4.

(21) Introduction whereas the right lobe is irrigated by the cranial and caudal pancreaticoduodenal arteries (branches from the gastroduodenal artery (cranial) and cranial mesenteric artery (caudal), respectively). Anastomoses are common in pancreatic circulation. Veins are parallel to the arteries and eventually drain into the portal vein. Although the islets constitute only 2-3% of the total pancreas mass, they receive around 20% of the blood flow (Iismaa et al., 2000). The pattern of blood flow through the islets appears to be specifically organized. Blood flows from ß to and then to cells (Bonner weir S., 1991) and presumably carries metabolic and hormonal information from the ß-cells to the mantle. The pancreatic islets are innervated by parasympatic cholinergic, sympathic adrenergic and various peptidergic nerves. The pancreas receives its extrinsic innervation from the celiac plexus.. 2. TYPE 1 DIABETES.. Diabetes mellitus is the most prevalent metabolic disorder, and is characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (Pickup, J.C. and Williams, G., 1994). Symptoms of marked hyperglycemia include polyuria, polydipsia, weight loss, polyphagia, and blurred vision. Impairment of growth and susceptibility to certain infections may also accompany chronic hyperglycemia with ketoacidosis or the nonketotic hyperosmolar syndrome (The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 1997). Diabetes mellitus is usually classified as type 1 or type 2 diabetes. Type 1 diabetes, previously encompassed by the terms insulin-dependent diabetes, which. 5.

(22) Introduction affects 5 to 10% of the diabetic population, typically appears during youth and results from the autoimmune destruction of the islet ß-cells within the pancreas (Atkinson and Maclaren, 1994). Markers of the immune destruction of the ß-cell include islet cell autoantibodies (ICAs), autoantobodies to insulin (IAAs), autoantibodies to glutamic acid decarboxylase (GAD65), and autoantibodies to the tyrosine phosphatases IA-2 and IA-2ß (Baekkeskov et al., 1982; Atkinson et al., 1986; Christie et al., 1992; Kaufman et al., 1992; Schmidli et al., 1994; Schott et al., 1994; Myers et al., 1995; Lan et al., 1996; Lu et al., 1996). One and usually more of these autoantibodies are present in 85-90% of individuals when fasting hyperglycemia is initially detected. Also, the disease has strong HLA associations, with linkage to DQA and B genes, and it is influenced by the DRB genes (Cantor et al., 1995; Huang et al., 1996). These HLA-DR/DQ alleles can be either predisposing or protective. In this form of diabetes, the rate of ß-cell destruction is quite variable, being rapid in some individuals (mainly infants and children) and slow in others (mainly adults) (Zimmet et al., 1994). Autoimmune destruction of ß-cells has multiple genetic predispositions and is also related to environmental factors that are still poorly defined. Although patients are rarely obese when they present this type of diabetes, the presence of obesity is not incompatible with the diagnosis. These patients are also prone to other immune disorders such as Grave’s disease, Hashimoto’s thyroiditis, Addison’s disease, vitiligo, and pernicious anemia. Some forms of type 1 diabetes have no known etiologies. Some of these patients have permanent insulinopenia and are rare prone to ketoacidosis, but have no evidence of autoimmunity. Although only a minority of patients with type 1 diabetes fall into this category, of those who do, most are of African and Asian origin. Individuals with this. 6.

(23) Introduction form of diabetes suffer from episodic ketoacidosis and exhibit varying degrees of insulin deficiency between episodes. This form of diabetes is strongly inherited, lacks immunological evidence for ß-cell autoimmunity, and is not HLA associated. There is an absolute, yet periodic, requirement for insulin replacement therapy in affected patients (Banerji and Lebovitz, 1989).. 3. CURRENT THERAPY FOR TYPE 1 DIABETES.. 3.1. Insulin replacement. Conventional treatment of type 1 diabetes patients consists in daily subcutaneous injections of recombinat insulin, which attempt to mimic the physiological levels of the hormone. However, glycemia is not always properly regulated, and chronic hyperglycemia leads to severe microvascular, macrovascular and neurological complications (Pickup, J.C. and Williams, G 1994). The Diabetes Control and Complications Trial Group compared the effect of intensive therapy, administered either with an external pump or by three or more daily insulin injections, with conventional treatment, consisting in one or two daily insulin injections, in the ability to prevent secondary complications (The Diabetes Control and Complications Trial Research Group, 1993). Intensive therapy of patients with type 1 diabetes delays the onset and slows the progression of clinically important retinopathy, nephropathy, and neuropathy, by a range of 35 to more than 70 percent. In contrast, the risk of severe hypoglycemia was three times higher with intensive therapy.. 7.

(24) Introduction 3.2. Pancreas or islet transplantation.. 3.2.1. Pancreas transplantation. Pancreas transplantation was first described in 1967 (Kelly et al., 1967), but initial pancreas graft and patient survival rates were dismal. A variety of factors, including advances in surgical techniques, immunosuppression, graft preservation techniques, methods of diagnosis and treatment of rejection, and management of common posttransplant complications, have led to significant improvements in graft and patient survival. As a result, the total number of pancreas transplant procedures reported to United Network of Organ Sharing (UNOS) and the International Pancreas Transplant Registry (IPTR) continued to increase, a total of 18,843 from December 1966 to October 2002, with most (13,951) performed in the United States (Gruessner and Sutherland, 2002). Pancreas transplantation is actually a group of procedures, each with particular characteristics that, in some cases, may have different, immediate, and possibly longterm complications and outcomes. In most cases, pancreas transplantation is performed in the setting of type 1 diabetes with end-stage renal disease (ESRD). The frequency of diabetes as the etiology of ESRD has doubled over the last decade so that diabetes, both type 1 and type 2, is now the most common cause of new ESRD (Collins et al., 2005). Because the longevity of patients with type 1 diabetes has increased, more individuals are at risk of developing ESRD, increasing the number of type 1 diabetes patients also eligible for combined or simultaneous pancreas-kidney transplant. Kidney transplant markedly improves patient survival in the diabetic ESRD patient compared with dialysis, especially when performed early (Ojo et al., 1998; Wolfe et al., 1999; MeierKriesche et al., 2000; Mange et al., 2001). Therefore, the impact of adding a pancreas. 8.

(25) Introduction graft, as with simultaneous pancreas-kidney transplant, should improve patient survival to that of kidney transplant alone. There are three main types of pancreas transplantation (Larsen, 2004): 1) simultaneous pancreas-kidney transplant, in which the pancreas and kidney are transplanted from the same deceased donor; 2) pancreas after-kidney transplant, in which a cadaveric, or deceased, donor pancreas transplant is performed after a previous, and different, living or deceased donor kidney transplant; and 3) pancreas transplant alone for the patient with type 1 diabetes who usually has severe, frequent hypoglycemia, but adequate kidney function. Pancreas transplant alone and pancreasafter-kidney transplant candidates must have stable, adequate kidney function at the time of transplant, as both the transplant operation and immunosuppression can otherwise cause an immediate further decline of renal function. Immediate complications that can occur with all types of pancreas transplant include rejection, thrombosis, pancreatitis, and infection.. 3.2.2. Islet transplantation. Islet transplantation has been investigated as a treatment for type 1 diabetes mellitus in selected patients with inadequate glucose control despite insulin therapy. However, the hope that such an approach would result in long-term freedom from the need for exogenous insulin has failed to materialize in practice until the Edmonton protocol described by Shapiro and collaborators in 2000 (Shapiro et al., 2000). Since 1990 to 2000, of the 267 allografts transplanted only 12.4 % have resulted in insulin independence for periods of more than one week, and only 8.2 % have done so for periods of more than one year. In the majority of these procedures, the regimen of immunosuppression consisted of antibody induction with an antilymphocyte globulin. 9.

(26) Introduction combined with cyclosporine, azathioprine, and glucocorticoids (Brendel et. al 1999). However, for any type of transplantation procedure, a balance is sought between efficacy and toxicity. To address this problem, the Edmonton group used a glucocorticoid-free immunosuppressive protocol that included sirolimus, lowdose tacrolimus and a monoclonal antibody against the interleukin-2 receptor (daclizumab) for a trial of islet transplantation alone in patients with type 1 diabetes (Shapiro et al., 2000). All patients quickly attained sustained insulin independence after transplantation. All recipients required islets from two donor pancreases, and one required a third transplant from two donors to achieve sustained insulin independence (Shapiro et al., 2000). A five-year follow up of the Edmonton group was reported recently (Ryan et al., 2005). Sixty-five patients received an islet transplant in Edmonton as of 1 November 2004. Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants, and 11 subjects had three transplants. In the completed patients, 5year follow-up reveals that the majority (approximately 80%) have C-peptide present post-islet transplant, but only a minority (approximately 10%) maintain insulin independence. The median duration of insulin independence was 15 months. In addition, islet transplantation relieved glucose instability and problems with hypoglycemia (Ryan et al., 2005). The results, though promising, still suggest the need for further progress in the availability of transplantable islets, improving islet engraftment, preserving islet function and reducing toxic immunosuppression. Therefore, the search for alternative sources of ß-cells and protection of these ß-cells upon transplantation are very active fields in diabetes research.. 10.

(27) Introduction 4. CELL THERAPY FOR TYPE 1 DIABETES. Diabetes mellitus has long been targeted, as yet unsuccessfully, as being curable with cell and/or gene therapy. The main hurdles have not only been the vector but also the lack of physiological regulation of the expressed insulin. Advances in understanding the developmental biology of ß-cells and the transcriptional cascade that drives it have enabled both in vivo and ex vivo gene therapy combined with cell therapy to be used in animal models of diabetes with success. The associated developments in the stem cell biology, immunology and gene transfer vectors have opened up further opportunities for gene therapy to be applied to target type 1 diabetes.. 4.1. Cell therapy for type 1 diabetes. The considerable manipulations that are required to convert non-ß-cells into efficient glucose-sensing, insulin-secreting cells have led investigators into considering means of expanding adult or neonatal ß-cells or of harnessing the developmental potential of islet precursors cells and of embryonal stem cells.. 4.2. Stem cells A stem cell is, by definition, the one cell capable of duplicating itself and resuming its undifferentiated status, while also originating progeny that can differentiate into one or more final products that are physiologically defined by their specific functions (Wagers and Weissman, 2004). Proceeding through the differentiation pathway, stem cells can be categorized as totipotent, pluripotent, multipotent, oligopotent, and unipotent, depending upon all their possibly reversible, progressively. 11.

(28) Introduction acquired characteristics (Fig. 2) (Wagers and Weissman, 2004).. Figure 2. Classification of stem cells based on their developmental potential according to Wagers and Weissman (Wagers and Weissman, 2004). Totipotent, able to give rise to all embryonic and extraembryonic cell types; pluripotent, able to give rise to all cell types of the embryo proper; multipotent, able to give rise to a subset of cell lineages; oligopotent, able to give rise to a restricted subset of cell lineages; unipotent, able to contribute only one mature cell type.. 4.2.1. Embryonic stem cells Embryonic stem cells (ESC) are pluripotent cell lines derived from the inner cell mass of blastocyst-stage embryos (Shamblott et al., 1998; Thomson et al., 1998; Cowan et al., 2004; Hwang et al., 2004) and their differentiation in culture may reproduce characteristics of early embryonic development. It has been shown that in vitro differentiation of mouse ESC can generate embryoid bodies, which, after selection for nestin-expression, can be stimulated to differentiate towards a ß-cell-like phenotype (Lumelsky et al., 2001). Variations in ESC-culture conditions can generate cells with properties of ß-cells (Kahan et al., 2003; Kania et al., 2003; Stoffel et al., 2004) and the expression of transcription factors, such as pax4 or pdx-1, have yield promising results differentiating ESCs into ß-like cells (Blyszczuk et al., 2003; Miyazaki et al., 2004). In these experiments, the early and uncontrolled introduction of transcription factors into ESC during in vitro differentiation did not yield encouraging results (Stoffel et al., 2004), whereas regulated expression of the introduced transcription factors might be more successful (Miyazaki et al., 2004). However, some doubt has been cast on whether ESC differentiation protocols truly yield cells that produce insulin, or cells that. 12.

(29) Introduction merely absorb insulin from the medium (Rajagopal et al., 2003). On the other hand, it has been demonstrated that differentiated cells do synthesize and release insulin, leading to reversal of diabetes in rodents after transplantation of ESC derived insulin-producing cells (Hori et al., 2002; Blyszczuk et al., 2003). Similarly, ESC genetically selected for insulin expression and injected into diabetic rats improved glucose control (Soria et al., 2001). However, to induce differentiation of human ESC in order to produce insulin particular culture conditions are needed (Assady et al., 2001; Segev et al., 2004). In this regard, human ESC cell culture techniques that do not require murine feeder cells have been developed, allowing for single species propagation of ESC and avoiding possible zoonotic infection of cells intended for clinical use (Moritoh et al., 2003). With the prospect of in vivo ESC transplantation, possible hazards and problems such as control of differentiation and teratoma formation, still remain to be overcome (Sipione et al., 2004). Nevertheless, existing ESC lines are far from being ideal for generating ß-cells and additional ESC lines continue to be generated (Cowan et al., 2004). Finally, in addition to scientific hurdles, ethical concerns about the use of ESC need to be addressed and resolved in the face of this powerful technology.. 4.2.2. Adult stem cells. 4.2.2.1. Adult stem cells isolated from the pancreas, liver or intestine. Progenitor cells resident in isolated pancreatic tissue might be able to give rise to endocrine islet cells. Human and rodent pancreatic-duct cells (Bonner-Weir et al., 2000; Ramiya et al., 2000) islet-derived cells (Teitelman, 1996; Zulewski et al., 2001) and exocrine tissue (Lipsett and Finegood, 2002) contain cells that can differentiate towards a pancreatic endocrine phenotype. These tissues, cultured and differentiated in vitro, have been transplanted and can reverse diabetes mellitus in rodents. Interestingly, it has. 13.

(30) Introduction been recently shown that non-endocrine epithelial cells obtained from the human pancreatic tissue discarded after islet isolation, can differentiate into insulin producing cells (Hao et al., 2006). However, to induce this differentiation co-transplantation with fetal human islet-like cell clusters under the kidney capsule of mice recipients was required. Therefore, a bone-fide pancreatic stem cell for ß-cell regeneration remains elusive. A genetic-marking study in mice casts doubt on the existence of any ß-cell progenitor cells and suggests that ß-cells regenerate only by proliferation of existing ßcells (Dor et al., 2004). In human beings, early immunological intervention to stop ßcell destruction during the development of type 1 diabetes mellitus allows recovery of pancreatic endocrine function (Herold et al., 2002; Glandt et al., 2003). This finding might in part be attributable to recovery in ß-cell mass by recruitment of local pancreatic or extrapancreatic progenitor cells that differentiate into cells and/or proliferation of remaining ß-cells during protection from immune-mediated destruction. Similarly, in NOD mice treated with Freud’s complete adjuvant, the immune response against ß-cells was blocked and this intervention allowed recovery from diabetes by increasing ß-cell mass (Kodama et al., 2003; Chong et al., 2006; Nishio et al., 2006; Suri et al., 2006).. 4.2.2.2. Bone marrow stem cells Bone marrow harbours cells that can become parenchymal cells after entering the liver, intestine, skin, lung, skeletal muscle, heart muscle, and central nervous system (Herzog et al., 2003) in rodent models and in human recipients of marrow or organ transplantation (Theise et al., 2000; Korbling et al., 2002). In rodents, haemopoietic organs harbour cells that can also differentiate into functional pancreatic endocrine cells (Ryu et al., 2001; Hess et al., 2003; Ianus et al., 2003; Kodama et al., 2003; Zorina et. 14.

(31) Introduction al., 2003; Kofman et al., 2004). For example, in one study, donor-derived cells were found in pancreatic islets of recipient mice 1-2 months after bone-marrow transplantation, (Ianus et al., 2003). These cells expressed insulin and genetic markers of ß-cells. When cultured, the donor-derived ß-like cells were shown to secrete insulin in response to glucose, and demonstrated intracellular calcium fluctuations similar to normal ß-cells. The authors demonstrated that 1–3% of the islet cells originated from the transplanted marrow in the studied period (Ianus et al., 2003). Since a marrowderived cell-type with pluripotential capacity to transdifferentiate into various phenotypes has been described (Jiang et al., 2002a), this or a similar cell type might be able to differentiate into pancreatic ß-cells. Similar transplantation experiments have been done in overtly diabetic mice whose ß-cells have been destroyed by streptozotocin (STZ)(Hess et al., 2003). After bone-marrow transplantation, blood glucose and insulin concentrations were normal, and survival of animals was better (Hess et al., 2003). In islets, marrow-derived cells were shown to have differentiated into endothelial cells and, more rarely, into insulin-expressing cells (Hess et al., 2003). In these experiments, endothelial engraftment was speculated to stimulate the proliferation of local pancreatic progenitors, leading in turn to the increased insulin-producing cell mass. Although these studies show the possibility of bone marrow transplantation as a therapeutic approach for ß-cell replacement, the immunological destruction of newly regenerated ß-cells in type 1 diabetes remains a problem.. Bone-marrow transplantation can be used to induce microchimerism. Thus, in non-obese diabetic (NOD) mice, an autoimmune model of type 1 diabetes, transplanted with marrow before development of autoimmune diabetes, chimerism prevented. 15.

(32) Introduction development of diabetes mellitus, presumably by mechanisms involving donor immunoregulatory cells preventing the host cells from becoming autoreactive against ß-cells. By contrast, in NOD mice that were already diabetic, induction of chimerism by sublethal or lethal irradiation followed by marrow transplantation did not result in recovery from diabetes (Zorina et al., 2003). However, when these diabetic transplantrecipients were kept normoglycaemic by insulin therapy, they ultimately recovered from diabetes. Analysis of pancreatic tissue showed increased proliferative activity and regeneration of ß-cells. Thus, marrow transplantation to induce immunological control plus maintenance of normoglycaemia allowed local or progenitor cells to proliferate as an adaptive response (Zorina et al., 2003). Transplantation of mesenchymal cells from the spleen combined with Freund’s complete adjuvant led to reversal of diabetes accompanied by regeneration of insulinproducing islets (Ryu et al., 2001) The transplanted splenic mesenchymal cells differentiated into ß-cells (Kodama et al., 2003). Thus splenic mesenchymal cells transplanted under certain conditions seem not only to keep immune destruction of islets in check, but also could transdifferentiate into pancreatic ß-cells. However, recent experiments demonstrated that restoration of b cell mass using this protocol was not due to transdifferentiation of splenocytes into b cells as the cause for b cell mass restoration (Chong et al., 2006; Nishio et al., 2006; Suri et al., 2006). Bone marrow cells can differentiate in vitro under controlled conditions into insulin expressing cells (Jiang et al., 2002a; Jahr and Bretzel, 2003) and such cells, transplanted under the kidney capsule of diabetic rodents, correct hyperglycemia. Removal of the grafted kidney returned the animals to a diabetic state (Oh et al., 2004) Cell fusion has been suggested as a mechanism of apparent adaption of bone-marrowderived cells into an extramedullary phenotype (Wagers et al., 2002; Kofman et al.,. 16.

(33) Introduction 2004) Studies involving pancreatic endocrine-cell differentiation from haemopoieticorgan derivatives largely rule out cell-fusion events as a mechanism of transdifferentiation (Ryu et al., 2001; Hess et al., 2003; Ianus et al., 2003; Kodama et al., 2003; Zorina et al., 2003; Hao et al., 2006). Bone marrow-derived extramedullary parenchymal tissue is not always found (Theise and Wilmut, 2003). Importantly, several groups found little if any transdifferentiation of bone marrow into ß-cells (Choi et al., 2003; Lechner et al., 2004; Taneera et al., 2006) or bone marrow derivation of intraislet endothelial cells in diabetic mice (Lechner et al., 2004). One group reported the generation of insulin-producing cells in liver, adipose tissue, spleen, and bone marrow in rodent models of diabetes mellitus (Kojima et al., 2004). Bone marrow transplantation showed that most if not all extrapancreatic insulin-producing cells derive from donor bone-marrow (Kojima et al., 2004).. 17.

(34) Introduction 5. GENE THERAPY.. 5.1. Introduction to gene therapy. The major goal of gene therapy is to introduce a functional gene in a target cell and restore protein production that is absent or deficient due to a genetic disorder. A key factor in the success of gene therapy is the development of gene delivery systems that are capable of efficient gene transfer in a broad variety of tissues, without causing any pathogenic effect. Over the years, a number of gene transfer vehicles have been developed that can roughly divided into two categories: non-viral and viral vectors. Non-viral vector gene delivery systems include direct injection of naked DNA and encapsulation of DNA with cationic lipids (liposomes) as delivery systems to introduce the genetic material into a target cell. Viral delivery systems exploit the capacity of viruses to enter the target cells, transfer the DNA to the cell nucleus and express proteins.. 5.2. Viral vectors. Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. The viral life cycle can be divided into two temporally distinct phases: infection and replication. Infection results in the introduction of the viral genome into the cell. This leads to an early phase of gene expression characterized by the appearance of viral regulatory products, followed by a late phase, when structural genes are expressed and assembly of new viral particles occurs. In the case of gene therapy vectors, the viral. 18.

(35) Introduction particles encapsulate a modified genome carrying a therapeutic gene cassette in place of the viral genome. Transduction is defined as the abortive (non-replicative or dead-end) infection that introduces this functional genetic information expressed from the recombinant vectors into the target cell (Fig. 3).. Figure 3. Transduction of the target cell. The vector particle containing the therapeutic gene sequences binds to a cell, generally through a receptor mediated process and then enters the cell, allowing the genome to enter the nucleus. The vector genome may go through complex processes but ends up as dsDNA that, depending on the vector, can persist as an episome or become integrated into the host genome. Expression of the therapeutic gene follows.. The relative concentration of vectors is measured as a titer expressed as the concentration of viral particles and/or the number of virions that are capable of transduction. The transducing particles usually represent a small percentage of total particles, and can vary between different preparations. Particle titer and an infectious or transducing titer are both important, because impurities and variations in infectious activity can influence efficacy, toxicity and immunogenicity.. 19.

(36) Introduction For gene therapy to be successful, an appropriate amount of a therapeutic gene must be delivered into the target tissue without substantial toxicity. Each viral vector system is characterized by an inherent set of properties that affect its suitability for specific gene therapy applications. For some disorders, long-term expression from a relatively small proportion of cells would be sufficient (for example, some genetic disorders), whereas other pathologies might require high, but transient, gene expression. Problems that may be observed with gene transfer vectors include acute toxicity from the infusion of foreign materials, cellular immune responses directed against the transduced cells, humoral immune responses against the therapeutic gene product and the potential for insertional mutagenesis by certain integrating vectors.. Viral vectors currently available for gene therapy are based on different viruses and can roughly be categorized into integrating and nonintegrating vectors. Vectors based on retroviruses, including lentivirus and foamy virus, have the ability to integrate their viral genome into the chromosomal DNA of the host cell, which will possibly achieve life-long gene expression. Wild type adeno-associated virus are integrating vectors, however, recombinant AAV vectors have lost this property because of the lack of viral proteins in the recombinant genome. Then, AAV can be essentially considered as a non-integrating vector although few cases of non-specific integration have been described (Nakai et al., 2003). Vectors based on adenovirus and herpes simplex virus type 1 (HSV-1) represent non-integrationg vectors. The non-integrating vectors deliver their genomes into the nucleus of the target cell, where they remain episomal.. 20.

(37) Introduction 5.2.1. Adenoviral vectors.. 5.2.1.1. Adenovirus structure and biology. Adenovirus was first isolated from human adenoid cells more than 50 years ago (Rowe et al., 1953). Since then, more than 100 different adenovirus species have been identified from various mammals, birds, and reptiles and all are characterized by a distinctive architecture and common chemical composition (Büchen-Osmond, 2003). Human adenoviruses are classified into six subgroups (A–F), which are further divided into serotypes based on their immunological properties. Serotype 2 (Ad2) and serotype 5 (Ad5) of subgroup C, which are the most extensively studied of the human adenoviruses, are found worldwide. The adenovirus virion is a nonenveloped icosahedral particle about 70–90 nm in size with an outer protein shell surrounding an inner nucleoprotein core (Fig. 4).. Figure 4. (A) Section of the adenovirus virion, indicating the relative orientations of the protein components and the viral DNA. The core and capsid proteins are listed separately (Stewart et al., 1993). (B) A 15-Å resolution three-dimensional cryo-EM image reconstruction of Ad5 viewed along the 3-fold axis of the icosahedral capsid The trimeric towers of hexon, the major coat protein, create the features visible on the facets. The fiber protrudes from the penton base at each 5-fold vertex. Only a short portion of the long fiber is visible; the rest is washed out when averaged by the reconstruction process.. 21.

(38) Introduction The facets of the virus capsid are composed primarily of trimers of the hexon protein, as well as a number of other minor components including protein IIIa (pIIIa), pVI, pVIII, and pIX. The capsid vertices consist of the penton base, which acts to anchor the fiber protein, the moiety responsible for primary attachment of virions to the cell surface. Adenovirus cores contain the viral DNA as well as pV, mu, and the histone-like protein pVII. The genome itself is a linear, double-stranded DNA that is approximately 36 kb long. Each end of the genome has an inverted terminal repeat (ITR) of 100–140 bp to which the terminal protein is covalently linked. Genes are encoded on both strands of the DNA in a series of overlapping transcription units (Fig. 5).. Figure 5. Map of the adenovirus genome and transcription units. The central, solid line represents the viral genome. Positions of the left and right ITRs, the packaging sequence (), the early transcription units (E1A, E1B, E2, E3, and E4), and the major late transcription unit (major late promoter [MLP], L1–L5) are shown. Arrows indicate the direction of transcription.. For all groups, except group B adenoviruses, initial attachment of virion particles to the cell surface occurs through binding of the fiber knob to the coxsackievirus B and adenovirus receptor (CAR) (Bergelson et al., 1997). After initial attachment to the cell surface, an exposed RGD protein motif on the penton base interacts with members of the v integrin family, triggering virus internalization by clathrin-dependent, receptor-mediated endocytosis (Stewart et al., 1997; Meier et al.,. 22.

(39) Introduction 2002). The first viral transcription unit to be expressed is E1A. As with almost all adenovirus transcription units, E1A produces multiple mRNA and protein products by way of differential mRNA processing. During infection, the E1A proteins function to trans-activate the other adenovirus early transcription units (E1B, E2, E3, and E4) and to induce the cell to enter S phase in order to create an environment optimal for virus replication (Berk, 1986). The E2 region encodes proteins necessary for replication of the viral genome: DNA polymerase, preterminal protein, and the 72-kDa single-stranded DNA-binding protein (de Jong et al., 2003). Products of the viral E3 region function to subvert the host immune response and allow persistence of infected cells (Gooding et al., 1991; Bruder et al., 1997; Bennett et al., 1999). The E4 transcription unit encodes a number of proteins that have been known to play a role in cell cycle control and transformation (Tauber and Dobner, 2001). Most adenovirus late genes are expressed from five regions, L1–L5, and are transcribed from one promoter, the major late promoter (MLP). These transcripts primarily encode structural proteins of the virus and other proteins involved in virion assembly. The packaging sequence itself is a series of seven repeats at the left end of the genome (Hearing et al., 1987) which mediates genome encapsidation (Grable and Hearing, 1990). Cell lysis and release of progeny virions occur approximately 30 hr postinfection in a process involving the E3-11.6K protein, also called the adenovirus death protein (ADP) (Tollefson et al., 1996). Several types of adenoviral vectors have been developed and extensively used in different gene therapy approaches.. 23.

(40) Introduction 5.2.1.2 First and second generation adenoviral vectors. The initial strategy used in the construction of adenovirus vectors was the replacement of the E1 region with the desired transgene. Since E1 region are necessary for activation of viral promoters and expression of both early and late genes, removal of the E1 coding sequence results in viruses that are severely impaired in their ability to replicate. This strategy give raise the so-called first generation (FG) adenoviral vectors. The ability to delete the E1 region is made possible by the existence of cell lines that provide these functions in trans. The classic cell line for this purpose is the 293 cell line, a human embryonic kidney-derived line that has been transformed by the adenovirus E1 region (Graham et al., 1977). The production of FG vectors was initially carried out by homologous recombination in mammalian cells between constructs carrying the left and right ends of the genome (Chinnadurai et al., 1979). However, this method proved to be inefficient and has prompted the development of techniques relying on standard cloning in bacteria and subsequent transfection of recombinant chromosomes into mammalian cells for virus production (Danthinne and Imperiale, 2000). Removal of the E1 region alone gives approximately 5.1 kb of cloning space without affecting viral titer and growth rate (Bett et al., 1993). In addition to E1 deletion, many of the first-generation vectors also contain a deletion in the E3 region, which is not essential for in vitro production of the vectors, in order to increase cloning capacity up to 8.2 kb. The main problem associated with the use of FG vectors is their stimulation of a cellular immune response, resulting in the destruction of transduced cells that are expressing therapeutic transgenes. Indeed, a number of early studies showed that administration of E1-deleted vectors to immune-competent animals results in only transient transgene expression (Yang et al., 1994a; Dai et al., 1995; Yang et al., 1995). It is theorized that the immune response is stimulated by low levels of replication that. 24.

(41) Introduction can occur even in the absence of the E1 genes. This idea is supported by findings that genome replication and late gene expression can occur from E1-deleted vectors in vivo (Yang et al., 1994a; Yang et al., 1994b). Therefore, the use of first-generation vectors is precluded for applications requiring short term gene expression such as cancer therapy and vaccination. To prevent the immune response generated by FG adenoviral vectors, a second generation was constructed by the removal of E2 and E4 coding sequences. These vectors provide larger capacity for transgene insertion and reduced immunity. However, the major drawback encountered during construction of these multiply deleted viruses is the need for isolation of cell lines expressing the missing functions in trans (Schaack et al., 1995; Amalfitano et al., 1996; Gorziglia et al., 1996; Amalfitano and Chamberlain, 1997) Nevertheless, the advantages of second generation Ad over FGAd remain controversial as some studies show them to be superior in terms of toxicity and longevity of transgene expression (Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al., 1994b; Goldman et al., 1995; Gao et al., 1996; Dedieu et al., 1997; Wang et al., 1997), whereas other studies do not (Fang et al., 1996; Morral et al., 1997; Lusky et al., 1998; O'Neal et al., 1998; Reddy et al., 2002).. 5.2.1.3. Helper dependent adenoviral vectors. Significant improvement in the safety and efficacy of Ad-based vectors came with the development of helper-dependent adenoviral vectors (HDAds, also referred to as gutless, gutted, fully deleted, or high-capacity) which are deleted of all viral coding sequences. Comparison of first and second generation vectors with Helper-dependent vectors genomes is shown in Fig. 6. HDAds retain the advantages of FGAds, including high-efficiency in vivo transduction and high-level transgene expression. However,. 25.

(42) Introduction owing to the absence of viral gene expression in transduced cells, these HDAds are able to mediate high-level, long-term transgene expression in the absence of chronic toxicity (Morral et al., 1998; Morsy et al., 1998; Schiedner et al., 1998; Morral et al., 1999; Balague et al., 2000; Cregan et al., 2000; Kim et al., 2001; Maione et al., 2001; Oka et al., 2001; Zou et al., 2001; Ehrhardt and Kay, 2002; Reddy et al., 2002) (Gilbert et al., 2002; Gilbert et al., 2003; Dudley et al., 2004; Fleury et al., 2004; Pastore et al., 2004; Wen et al., 2004; Brunetti-Pierri et al., 2005; Mian et al., 2005; Toietta et al., 2005). Other advantages of these vectors include lack of germ line transmission and insertional mutagenesis because the vector genome exists episomally in transduced cells. Moreover, the deletion of the viral sequences permits a large cloning capacity of 37 kb, allowing for the delivery of whole genomic loci, multiple transgenes, and large cis acting elements to enhance, prolong, and regulate transgene expression.. Figure 6. Genome structure of first-generation, second-generation, and helper-dependent vectors. Regions that have been deleted are indicated by open boxes.. Because HDAds are deleted of all viral coding sequences, a helper virus is required for their propagation. The first efficient method for generating HDAds was the Cre–loxP system developed by Parks and coworkers in 1996 (Parks et al., 1996) (Fig.. 26.

(43) Introduction 7). In this system the HDAd genome is first constructed in a bacterial plasmid. Minimally, the HDAd genome contains the expression cassette of interest and 500 bp of cis-acting Ad sequences necessary for vector DNA replication (inverted terminal repeats, ITRs) and packaging ( ). Because efficient packaging into the Ad capsid requires a genome size between 27.7kb (Parks and Graham, 1997) and 38kb (Bett et al., 1993), “stuffer” DNA is usually included in HDAds. To generate the HDAd viral particles, the plasmid is first digested with the appropriate restriction enzyme to liberate the HDAd genome from the bacterial plasmid sequences. 293 cells expressing the sitespecific recombinase Cre are then transfected with the linearized HDAd genome and subsequently infected with the helper virus. The helper virus, bears a packaging signal flanked by loxP sites, the target sequence for Cre, and thus after infection of 293Cre cells the packaging signal is excised from the helper viral genome by Cre-mediated sitespecific recombination between the loxP sites. This renders the helper viral genome unpackageable, but still able to undergo DNA replication and thus trans-complement the replication and encapsidation of the HDAd genome. The titer of the HDAd is amplified by serial coinfection of 293Cre cells with the HDAd and the helper virus, and the HDAd is finally purified by CsCl ultracentrifugation.. 27.

(44) Introduction. Figure 7. The Cre–loxP system for producing HDAds. The HDAd contains only 500 bp of cis-acting Ad sequences required for DNA replication (ITRs) and packaging ( ); the remainder of the genome consists of the desired transgene and non-Ad stuffer sequences. The HDAd genome is constructed as a bacterial plasmid (pHDAd) and is liberated by restriction enzyme digestion (e.g., PmeI). To rescue the HDAd, the liberated genome is transfected into 293Cre cells and infected with a helper virus bearing a packaging signal ( ) flanked by loxP sites. Cre-mediated excision of ) renders the helper virus genome unpackageable, but still able to replicate and provide all of the necessary trans-acting factors for propagation of the HDAd. The titer of the HDAd is increased by serial coinfections (called passages) of 293Cre cells with the HDAd and the helper virus. Finally, the HDAd is purified by CsCl ultracentrifugation.(Parks et al., 1996). Major hurdless in the gutless vector generation include low efficiency in the production methods, genome instability, generation of replication competent particles and helper virus contamination. Since the original Cre-lox system was described others systems have been generated to circumvent these limitations ((Palmer and Ng, 2003; Sakhuja et al., 2003; Nakano et al., 2005; Oka and Chan, 2005; Shi et al., 2006)). A production system composed of a suspension-adapted producer cell line that expressed. 28.

(45) Introduction high levels of Cre, a helper virus resistant to mutation, and refined protocols led to production of titers >1013 viral particles and with exceedingly low helper virus contamination of 0.4–0.1% without relying on CsCl purification and 0.02–0.01% after CsCl purification (Palmer and Ng, 2003). Encouraging results have been obtained with HDAd in several animal model of disease. These vectors have been used extensively for liver-directed gene therapy because of their ability of transduce hepatocytes after systemic intravenous administration. HDAd-mediated gene therapy in baboons using vectors carrying the human 1-antitrypsin (hAAT) gene, showed expression of hAAT for more than 1 year in two of the three animals (Morral et al., 1999). Many other studies have exploited the advantages of HDAd for hepatic transduction (Ehrhardt and Kay, 2002; Belalcazar et al., 2003; Kojima et al., 2003; Mian et al., 2004; Wang et al., 2004b; Toietta et al., 2005). In addition to the liver, gene transfer to other tissues such as the brain (Zou et al., 2001), eye (Kreppel et al., 2002b; Oshima et al., 2004), muscle (Chen et al., 1997; Chen et al., 1999; Jiang et al., 2001; Maione et al., 2001; Gilbert et al., 2002; Gilchrist et al., 2002; Jiang et al., 2002b; Gilbert et al., 2003; Fleury et al., 2004; Jiang et al., 2004), and lung (Toietta et al., 2003; Koehler et al., 2006) has been described using these vectors.. 5.2.2. Adeno-associated viral vectors. Adeno-asociated virus (AAV) was originally identified as a contaminant of adenovirus cultures. AAV is a member of the dependovirus genus of the family Parvoviridae. Like other parvoviruses, AAV exists as a non-enveloped icosahedral particle with a diameter of approximately 20 nm. AAV has never been shown to cause any human disease, despite a high seroprevalence rate. Multiple serotypes of AAV have. 29.

(46) Introduction been identified, including human serotypes 1,2,3 and 5, simian serotype 4, as well as bovine, canine and avian AAV (Blacklow, 1988). However, since each serotype could have different cell tropism, many efforts are focused in the isolation and characterization of new serotypes to look for a tissue-specific serotype to be used as gene transfer vector. Thus, AAV serotype 6 was chracterized in 1999 and appears to be a recombinant between AAV1 and AAV2 (Xiao et al., 1999. Later, four new serotypes were isolated from nonhuman primates AAV7, AAV8, AAV9 and rh10 [Gao, 2002 #684; Gao et al., 2002). The AAV2 genome has been cloned (Samulski et al., 1982; Laughlin et al., 1983), sequenced (Srivastava et al., 1983), and characterized in detail, and this is the most widely use for the generation of recombinant vectors. The termini consist of 145nucleotide long ITRs. The ITRs contain all cis-acting functions required for DNA replication, packaging integration, and subsequent excision and rescue (Samulski et al., 1989; Srivastava et al., 1989). Internal to the ITR are two viral genes; rep, which encodes functions required for replication, and cap, which encodes structural proteins of the capsid. A total of four Rep proteins (Rep78, Rep68, Rep52, and Rep40) and three cap proteins (VP1, VP2, and VP3) are generated by alternative splicing. The AAV have the tendency to integrate within a specific region of human chromosome 19, the AAVS1 site (Kotin et al., 1990; Kotin et al., 1991; Samulski et al., 1991; Kotin et al., 1992; Samulski, 1993). The mechanism for integration may involve the Rep68 protein (Weitzman et al., 1994). Construction of AAV recombinant vectors can be done by several methods; however, five elements are generally required: (a) cells permissive for AAV replication (e.g. 293 cells), (b) a helper virus (e.g. adenovirus, or a plasmid including the adenovirus sequences), (c) a recombinant AAV vector of 5 kb or less, including ITRs. 30.

(47) Introduction flanking the transgene and any promoter, enhancer, or polyadenylation elements, (d) a source of rep proteins, and (e) a source of capsid proteins. These elements can be supplied by co-transfection two or three plasmids. Cells must be then be lysed by serial freeze-thaw or other physical methods to release the packaged virions. Packaged AAV vector can then be separated from cellular debris by CsCl gradient ultracentrifugation. The principal advantage of AAV over other DNA virus, such as adenovirus, is the lack of any viral coding sequence within the vector, which prevents transduced cells from being recognized and rejected by the immune systems. In addition, AAV is also efficient at cell entry and tends to persist in cells over long periods of time. Although the low immune reponse was true in animal models, it has been shown that AAV vectors could trigger an immune response in humans (Manno et al., 2006).. 6. GENE THERAPY FOR TYPE 1 DIABETES.. Limitions of the current therapy of insulin injections and islet transplantation can be potentially circumvented with gene therapy. Indeed, advances in cell and gene therapy have rekindled the possibility of not only ameliorating, but also potentially curing diabetes. Gene therapy strategies for diabetes can broadly be divided into three categories: preventive, adjunctive and curative. While preventive and adjunctive strategies primarily address the autoimmune pathogenesis of type 1 diabetes and the immune response to islet grafts post-transplantation, respectively, curative strategies involve restoring insulin production and secretion by effecting insulin transgene expression or by inducing islet neogenesis.. 31.

(48) Introduction 6.1. Gene therapies aimed at reducing immune reactions or inducing immune tolerance. Primary prevention of type 1 diabetes involves identifying patients at risk and instituting measures to prevent the autoimmune destruction of ß-cells such as immunomodulatory gene therapy. This has been shown to work in rodent models (Jun et al., 2002; Flodstrom-Tullberg et al., 2003; Goudy et al., 2003; Steptoe et al., 2003; Jin et al., 2004; Song et al., 2004). These strategies were aimed at inducing tolerance or hyporesponsiveness to islet autoantigens by either increasing tolerance-inducing regulatory T lymphocytes (IL-10 gene therapy (Goudy et al., 2003); by suppressing reactive T lymphocytes (immune therapy using recombinant vaccinia virus expressing glutamic acid decarboxylase (Jun et al., 2002); by targeting T lymphocytes for apoptosis (Jin et al., 2004); by inducing tolerance in ß-cells (by overexpression of suppressor of cytokine signaling-1 (SOCS-1) (Flodstrom-Tullberg et al., 2003); or by inducing antigen-presenting cells to express proinsulin, an autoantigen (Steptoe et al., 2003). However, the translation of the animal experiments to human trials is constrained by the lack of markers with acceptable positive and negative predictive values to unequivocally identify and select appropiate patients who are at risk for preventive therapy. Yet, conceptually, this is the area that will need to be explored if our ultimate goal is the primary prevention of type 1 diabetes. Preventive strategies have currently focused on secondary prevention, that is, measures to prevent the further loss of ß-cells once diabetes has set in. These are instituted optimally at diagnosis or in the honeymoon period following the initial acute presentation as often seen in type I diabetes (Herold et al., 2002; Kodama et al., 2003; Keymeulen et al., 2005; Chong et al., 2006; Nishio et al., 2006; Suri et al., 2006).. 32.

(49) Introduction 6.2. Genetic manipulation of extra-pancreatic cells to produce insulin. There have been substantial efforts to engineer glucose-responsive insulin secretion into cells other than ß-cells. For instance, gut K cells of the mouse were induced to produce human insulin by transfecting them with the human insulin gene linked to the 5’-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP) (Cheung et al., 2000). Rodent-liver stem cells and human fetal-liver cells have also been differentiated in vitro into insulin-secreting cells by introduction of ß-cell-specific genes. These experiments demonstrating differentiation to ß-like cells from adenovirus-transduced murine hepatocytes with the Pdx1 gene, alone (Ferber et al., 2000) or in combination with NeuroD (Kojima et al., 2003), attracted significant attention and has inspired new hope. Importantly, tranduction of human hepatocytes with pdx1 gave similar results and insulin production was also observed (Zalzman et al., 2003; Sapir et al., 2005; Zalzman et al., 2005). Sufficient levels of insulin were secreted to satisfy the needs of a diabetic mouse, which became, and remained, steadily euglycemic after treatment with pdx1 transduced cells.. 6.3. Gene transfer to the pancreas and/or ß-cells and its applications. Efficient and stable gene transfer to pancreatic cells is required for pursuing gene therapy strategies for ß-cell expansion and modification. To genetically engineer the endocrine pancreas the selection of both the vector and the route of administration are key. While several vectors can be used to transfer foreign genes to pancreatic islets and to pancreatic ß-cell lines in vitro, few attempts have been assayed to target the islets in vivo. The shortage of islets donors, together with the need of more than one donor per each recipient have increase the interest in genetic manipulations of the islets to avoid. 33.